The overall objective is to provide critical information on the physiology and biochemistry of sperm interactions with the egg investments (cumulus oophorus (CO) and zona pellucida (ZP)). Specific aims are (1) to determine where the fertilizing spermatozoon undergoes the acrosome reaction (AR) in relation to penetration of the egg investments. This question will be studied using video-micrography to record acrosomal changes (AR) in sperm as they penetrate egg investments in vitro. (2) To elucidate the functional significance and relative roles of the CO and ZP in stimulating the AR in fertilizing sperm. This will be done using a highly defined in vitro fertilization procedure. (3) To characterize the chemical nature of the acrosome reaction-inducing factor (ARIF) found in the gamete interactions at low sperm:egg ratios in order to probe the penetration of intact CO in vitro; (b) examination of the functional roles of egg investments in supporting sperm fertilizing ability; (c) investigation of the functional significance for AR-induction of the ZP separately from that of the CO; and (d) evaluation of the possible potentiation of ZP-ARIF by CO-ARIF. In addition, (e) we are able to capacitate spermatozoa under completely chemically-defined conditions, i.e., in the absence of biologically-active proteins such as serum albumin, which can confound attempts to examine naturally-occurring sources of ARIF. Taken together, the experiments will fill in some fundamental gaps in present knowledge about the interactions of the egg investments with spermatozoa, with particular emphasis on their roles in regulating sperm fertilizing ability.